We originally proposed that ring-opened metabolites of benzene may contribute significantly to its hematotoxicity. Consistent with this hypothesis, our work in this field initially demonstrated that benzene is metabolized in vitro to trans,trans-muconaldehyde (MUC), a reactive six carbon diene dialdehyde, and that MUC is toxic to the bone marrow in a manner similar to benzene. It has recently become apparent that benzene hematotoxicity is a complex process which most likely involves interaction between several intermediates which operate by more than one mechanism to produce more than one biological effect. Our own studies showed that MUC and hydroquinone (HQ) are a particularly potent combination in causing bone marrow damage, suggesting that the involvement of ring-opened metabolites in benzene toxicity may be related to their action in combination with other benzene metabolites. The mechanism of this interaction is not known. Muconaldehyde is a strong electrophile which readily reacts with DNA, proteins, and glutathione (GSH). The chemical reactivity of MUC as an alkylating agent, crosslinking agent and thiol depletor may provide the basis for molecular interactions which lead to bone marrow toxicity. The present application focusses on (i) mechanistic studies to probe several biological interactions of MUC which could be relevant to its role in benzene toxicity and carcinogenicity and (ii) studies on the demonstration that MUC is formed from benzene in vivo. The specific aims are: 1. To test the hypothesis that the synergistic hematotoxic interaction between MUC and HQ is a consequence of enhanced oxidative damage as a result of increased levels of toxic reactive oxygen species (ROS) and diminished antioxidant defenses in bone marrow cells. Transgenic mice with overexpressed levels of the antioxidant enzymes CuZn-superoxide dismutase (CuZnSOD) and glutathione peroxidase (GSH-Px) and their normal littermates will be used to determine (i) whether modulation of antioxidant status affects the hematotoxic response to benzene and MUC/HQ coadministration and (ii) whether bone marrow cell ROS levels, DNA oxidation and soluble thiol (GSH) levels in mice treated as described above change significantly and in a manner consistent with our hypothesis. 2. To test the hypothesis that DNA-protein crosslinking by MUC is in part responsible for the hematotoxicity of benzene by determining whether administration of MUC and benzene leads to significant increases in DNA- protein crosslinks (DNA PC) in bone marrow cells of CD-1 mice. 3. To assess the formation of MUC-GSH and MUC-serum albumin adducts in vivo in order to provide an assay of MUC formation and use it in studies on the correlation of benzene toxicity with MUC formation.